Dithiaarsanes induce oxidative stress-mediated apoptosis in HL-60 cells by selectively targeting thioredoxin reductase

Article abstract of DOI:10.1021/jm500221p

The selenoprotein thioredoxin reductase (TrxR) plays a pivotal role in regulating cellular redox homeostasis and has attracted increasing attention as a promising anticancer drug target. We report here that 2-(4-aminophenyl)-1,3,2- dithiarsinane (PAO-PDT, 4), a potent and highly selective small molecule inhibitor of TrxR, stoichiometrically binds to the C-terminal selenocysteine/cysteine pair in the enzyme in vitro and induces oxidative stress-mediated apoptosis in HL-60 cells. The molecular action of 4 in cells involves inhibition of TrxR, elevation of reactive oxygen species, depletion of cellular thiols, and activation of caspase-3. Knockdown of TrxR sensitizes the cells to 4 treatment, whereas overexpression of the functional enzyme alleviates the cytotoxicity, providing physiological relevance for targeting TrxR by 4 in cells. The simplicity of the structure and the presence of an easily manipulated amine group will facilitate the further development of 4 as a potential cancer chemotherapeutic agent.

Full text of DOI:10.1021/jm500221p

Article
pubs.acs.org/jmc
Dithiaarsanes Induce Oxidative Stress-Mediated Apoptosis in HL-60
Cells by Selectively Targeting Thioredoxin Reductase
Yaping Liu, Dongzhu Duan, Juan Yao, Baoxin Zhang, Shoujiao Peng, HuiLong Ma, Yanlin Song,
and Jianguo Fang*
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University,
Lanzhou, Gansu 730000, China
S
* Supporting Information
ABSTRACT: The selenoprotein thioredoxin reductase (TrxR)
plays a pivotal role in regulating cellular redox homeostasis and
has attracted increasing attention as a promising anticancer drug
target. We report here that 2-(4-aminophenyl)-1,3,2-dithiarsinane
(PAO−PDT, 4), a potent and highly selective small molecule
inhibitor of TrxR, stoichiometrically binds to the C-terminal
selenocysteine/cysteine pair in the enzyme in vitro and induces
oxidative stress-mediated apoptosis in HL-60 cells. The molecular
action of 4 in cells involves inhibition of TrxR, elevation of
reactive oxygen species, depletion of cellular thiols, and activation
of caspase-3. Knockdown of TrxR sensitizes the cells to 4 treatment, whereas overexpression of the functional enzyme alleviates the
cytotoxicity, providing physiological relevance for targeting TrxR by 4 in cells. The simplicity of the structure and the presence of an
easily manipulated amine group will facilitate the further development of 4 as a potential cancer chemotherapeutic agent.
isoforms within cells, TrxR1 and TrxR2 have similar structures
and share the same catalytic mechanism.12 Compared to those
from bacteria, mammalian TrxRs are large selenocysteine (Sec)-
containing proteins with a uniform C-terminal sequence of -Gly-
Cys-Sec-Gly, which is required for the activity of the enzyme.13
The chemical property of Sec (selenol) resembles, but is generally
more reactive than, that of Cys (thiol). Mammalian TrxR has
broad substrate specificity, but it is the only known enzyme to
reduce Trx in vivo. It was initially considered that TrxR maintains
reduced Trx pools for ribonucleotide reductase in DNA syn-
thesis14 as well as for many antioxidant enzymes such as peroxi-
redoxins15 and methionine sulfoxide reductases.16,17 It is becoming
increasingly clear that TrxR also plays critical roles in tumori-
genesis, and accumulating studies suggest that TrxR might be a
promising anticancer drug target.18−20 Thus, expanding attention
has been paid to the development and discovery of TrxR inhibitors
as potential chemotherapeutic agents during recent years.21−25
Arsenic has two biologically important oxidation states,
As(III) and As(V). Compared to the relatively inert property of
As(V), As(III) easily reacts with closely spaced thiols, yielding
stable cyclic dithioarsinite complexes in which both sulfur atoms
are bound to arsenic. Most of the effects of arsenic compounds
in cells have been through their ability to bind to thiols in
cysteine residues.26 Proteins whose function is dependent on
one or both of the cysteine thiols are thus inactivated by the
trivalent arsenicals. Also, many protein-labeling agents have
been developed on the basis of the high affinity of trivalent
1. INTRODUCTION
Arsenic is a semimetal or metalloid element widely distributed on
earth. The majority of arsenic is found in minerals in conjunction
with sulfur or metals. Arsenical-based molecules have been used
as therapeutic agents for many centuries for various ailments
such as psoriasis, syphilis, leukemia, and rheumatism.1−3
However, concerns about their toxicity eventually led to their
abandonment for the treatment of cancer before the clinical
discovery of the favorable efficacy of arsenic trioxide (ATO,
As2O3), which led to remission in patients with acute
promyelocytic leukemia (APL) in the 1980s.4 This old inorganic
compound has achieved remarkable clinical success and has been
validated as a front-line drug in the treatment of a number of
malignant diseases, especially APL.5 Organic arsenicals consist of
an arsenic atom linked covalently to a carbon atom. The unique
bonding property of carbon atom gives the diverse structure of
organoarsenicals. Besides their structural diversity, organo-
arsenicals are often more stable and less toxic and have better
bioavailability than inorganic arsenicals. Inspired by the success
of ATO, many synthetic organoarsenicals or hybrid arsenics are
currently under investigation for use in cancer treatment.6−9
The thioredoxin system, composed of thioredoxin reductase
(TrxR), thioredoxin (Trx), and NADPH, is a highly conserved
system and plays a crucial role in regulating a wide variety of
redox signaling pathways involved in cell proliferation and death,
transcription, DNA repair, angiogenesis, and embryogenesis.10,11
Two major isoforms of TrxR/Trx are present in different
intracellular organelles: TrxR1/Trx1 are predominantly localized
in the cytosol and nucleus, and TrxR2/Trx2 are mainly localized
within mitochondria. Despite the different localizations of the
Received: February 10, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
arsenicals with vicinal dithiols.27−30 Because the C-terminal
redox center, -Gly-Cys-Sec-Gly, is indispensable for the activity
of TrxR and because ATO was reported to be a potent inhibitor
of TrxR,31 we reasoned that trivalent arsenicals might be potent
inhibitors of TrxR and thus might be potential cancer
chemotherapeutic agents. As part of our continuing interest in
discovering and developing novel small molecule regulators of
cellular redox systems,21,32−36 we report herein the synthesis,
biological evaluation, and mechanism of action of phenylarsenic
oxide derivatives (Figure 1). 2-(4-Aminophenyl)-1,3,2-dithiarsinane
(PAO−PDT, 4) stoichiometrically binds to the C-terminal
selenocysteine/cysteine pair in TrxR in vitro. The inhibition of
TrxR by 4 is highly selective because other related enzymes, such
as the Sec-to-Cys mutant of TrxR (U498C TrxR), glutathione
reductase (GR), glutathione peroxidase (GPx), and Trx, are not
affected by 4. TrxR inhibited by 4 is still functional as a pro-
oxidant NADPH oxidase for the constant generation of reactive
oxygen species (ROS). Compound 4 displays its highest
cytotoxicity toward human promyelocytic leukemia HL-60 cells
among all tested cell lines. Further mechanistic study indicates
that the cellular action of 4 involves the inhibition of TrxR,
accumulation of ROS, depletion of cellular thiols, and induction
of apoptosis. Overexpression of functional TrxR confers cyto-
protection from 4, whereas knockdown of the enzyme enhances
the cytotoxicity, demonstrating the physiological significance of
targeting TrxR by 4 in cells. We expect that 4 could be a novel
cancer chemotherapeutic agent for further development.
2. RESULTS AND DISCUSSION
Figure 1. Chemical structures of organoarsenical compounds.
2.1. Chemical Synthesis. The dithiaarsanes were synthe-
sized as previously described37,38 via coupling the appropriate
dithiols with 4-aminophenylarsenoxide (PAO, 2), which was
obtained by the reduction of 4-aminophenylarsonic acid
(PAA).38,39 The dithiol ligands, ethane-1,2-dithiol (EDT) and
propane-1,3-dithiol (PDT), react with 2, affording 2-(4-
aminophenyl)-1,3,2-dithiarsolane (PAO−EDT, 3) and 4,
respectively. Further acetylation of 3 and 4 furnishes the cor-
responding acetylated compounds, 2-(4-acetamidophenyl)-
1,3,2-dithiarsolane (APAO−EDT, 5) and 2-(4-acetamidophenyl)-
1,3,2-dithiarsinane (APAO−PDT, 6). The synthetic route is
illustrated in Scheme 1. The structures formed between trivalent
arsenicals and dithiols are markedly more stable than that of the
noncyclic products formed with monothiols. Indeed, our attempt
to prepare acyclic dithiol arsenites from 2 and monothiols failed
a
Scheme 1. Synthesis of Organoarsenical Compounds
a
(a) MeOH, HCl, KI, SO2, rt, 30 min; (b) 10% NH4OH, rt, 56%; (c)
MeOH, RSH, reflux, 70%; (d) DMF, CH3COOH, EDCI, HOBT, 70%.
a
Scheme 2. Synthesis of PAO−Nap (12) and PAO−PDT−Nap (13)
a
(a) Boc2O/1,4-diaxane, rt, 90%; (b) Et3N/ClCO2Et/CH3CN/NH4OH, rt, 50%; (c) TFA, rt, 60%; (d) AcOH/HNO3, rt, 63%; (e) Na2Cr2O7/
AcOH, reflux, 76%; (f) SnCl2·2H2O/HCl/EtOH, reflux, 79%; (g) DMF, 100°C, 33%; (h) PDT/EtOH, reflux, 86%.
B
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Scheme 3. Preparation of Immobilized Organoarsenical Compounds
because the acyclic compounds were unstable and spontaneously
2.3. Selective Inhibition of TrxR in Vitro. Trivalent
decomposed to the starting materials.37 The fluorescence-labeled
arsenicals easily react with closely spaced protein thiols. As the
arsenic atom in 4 is already bound with PDT, we speculated
that its cytotoxicity might be caused by the replacement of
PDT by more reactive ligands present in the cells. In this sense,
TrxR might be a candidate for interaction with 4 for the
following reasons: (1) TrxR contains a highly reactive selenol
group, (2) the enzyme has a C-terminal sequence of -Gly-Cys-
Sec-Gly, providing a closely spaced selenol/thiol pair, and (3)
this selenol/thiol pair is accessible because it is exposed on the
surface of the enzyme. Indeed, 4 efficiently inhibits TrxR
(Figure 2A). Next, we compared the inhibition potency of 4
organoarsenical compounds were constructed by attaching a
naphthalimide fluorophore using aminocaproic acid as a linker
(Scheme 2).29 The immobilized 4 was prepared by conjugation
of the target molecule to sepharose 4B using 1,4-butanediol
diglycidyl ether as a linker (Scheme 3).40 The detailed synthetic
procedures and characterization of 2 and its various derivatives
are presented in the Supporting Information. The purity of the
synthesized compounds was determined by HPLC. Except for
the two labeling reagents, 12 (95.9% purity) and 13 (93.6%
purity), all other compounds have purities >98% (Table S1).
2.2. Cytotoxicity Screening. Initial cytotoxicity screening
is summarized in Table 1. PAA, a pentavalent arsenic
Table 1. Sensitivity of Different Types of Cancer Cell Lines
a
to Arsenic Compounds
cell lines
HL-60
7721
48 h
HeLa
48 h
HepG2
48 h
compounds
48 h
>10
72 h
>10
PAA
>10
>10
>10
>10
>10
>10
>10
>10
>10
2
6.2 1.5
2.5 0.6
0.7 0.1
2.5 0.3
0.8 0.4
6.5 0.8
5.1 1.1
1.7 0.4
0.6 0.1
2.0 0.2
0.7 0.2
5.7 1.3
9.4 0.2
9.9 1.9
2.8 1.0
9.0 1.3
3.4 0.9
>10
7.7 0.5
6.1 1.5
3.4 0.2
>10
3
4
5
6
4.6 0.9
>10
ATO
a
The data (IC50) were obtained by the MTT assay and are expressed
as the mean SD of three independent experiments.
Figure 2. Selectively targeting TrxR by 4 in vitro. (A) Inhibition of WT
TrxR, U498C TrxR, and GR by 4. NADPH-reduced recombinant rat
TrxR (80 nM), U498C TrxR1 (1.0 μM), and GR (0.25 U/mL) were
incubated with the indicated concentrations of 4 for 30 min at room
temperature, and the enzyme’s activity was determined. All activity is
expressed as the percentage of the control. Data are expressed as the
mean SE of three independent experiments. (B) Correlation of the
IC50 values of 4 and the TrxR concentrations. NADPH-reduced
recombinant rat TrxR (40, 80, 120, and 160 nM) was incubated with
different concentrations of 4 for 30 min at room temperature, and the
enzyme activity was determined by the DTNB assay. (C) Labeling of
different proteins by 12 and 13. The reduced proteins were incubated
with 12 or 13 for 30 min at room temperature. The samples were
separated by SDS-PAGE. Top: Fluorescence imaging of the gel;
Bottom: Commassie Blue (C. B.) staining of the gel. (D) Loss of free
thiol groups in Trx after treatment with 4. The remaining free -SH
groups in the reduced Trx after treatment with 2 or 4 for 0.5 h at 37 °C
were determined by DTNB titration. Data are expressed as the
mean SE of three independent experiments.
compound, shows the least toxicity, whereas other trivalent
arsenicals display high potency. This result agrees with previous
results that indicated that pentavalent arsenicals are usually less
toxic.41,42 In addition, derivatization of 2 with dithiol ligands to
cyclic dithiaarsanes 3 and 4 further improves the potency. Acetyla-
tion of the amino group, yielding 5 and 6, does not significantly
alter the activity. It appears that the six-membered dithiaarsanes (4
and 6) are more active than the five-membered ones (3 and 5).
The most potent dithiaarsane, 4, had a concentration causing half
inhibition of cell proliferation (IC50) of 0.7 and 0.6 μM after treat-
ment of HL-60 cells for 48 and 72 h, respectively. Furthermore, 4
shows less toxicity toward two normal cell lines, L02 and HEK
293T cells, with IC50 values of 10.2 and 2.7 μM, respectively
(Figure S1). Collectively, the results demonstrate that 4 displays
selective cytotoxicity toward HL-60 cells with high potency. Thus,
follow-up studies were mainly focused on compound 4.
C
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 3. Interaction of 4 and TrxR in cells. (A) Inhibition of TrxR activity in HL-60 cells by 2 and 4. Data are expressed as the mean SE of three
experiments. (B) Imaging TrxR activity in living HL-60 cells. HL-60 cells were treated with the indicated concentrations of 4 for 8 h followed by
further treatment with TRFS-green (10 μM) for 4 h. Phase-contrast (top) and fluorescence (bottom) images were acquired by fluorescence
microscopy (Leica DMI4000). (C) Quantification of relative fluorescence intensity in individual cells. Ten cells were selected randomly, and the
relative fluorescence intensity in individual cells in panel B was quantified using the Leica Qwin software accompanied by the Leica microscope. Data
are expressed as the mean SE. (D) Compound 4 binds to cellular TrxR1. The HL-60 lysate was incubated with Seph−PAO−PDT or A-Seph on a
rotator mixer for 30 min at room temperature. After centrifugation, the solution was removed, and the sepharose was washed and eluted with DMPS.
The eluent was analyzed by western blotting (lane b). Lane a: positive control using HL-60 cell lysate for western blotting. Lane c: control using
A-Seph instead of Seph−PAO−PDT. (E) Upregulation of TrxR1 protein expression in HL-60 cells. Cells were treated with the indicated
concentrations of 4 for 12 and 24 h, and the cell extracts were prepared and analyzed by western blotting with an antibody against TrxR1. Actin was
used as a loading control.
toward TrxR, GR, and U498C TrxR, where Sec498 was
replaced by Cys (Figure 2A). Compound 4 effectively inhibits
TrxR with an IC50 value around 36 nM, whereas it exhibits
very weak inhibition of GR (IC50 ≫ 10 μM) and even less
of an effect on U498C TrxR. The interaction of 4 with GPx,
a Sec-containing enzyme, was also investigated. No apparent
inhibition of GPx was observed, even in the presence of 10 μM
4 (Figure S2). Analysis of the IC50 value of 4 (∼36 nM) and
the TrxR concentration (80 nM) suggest the binding of 4 to
the enzyme in a 1:1 ration. We further determined the IC50
values of 4 toward varying concentrations of TrxR (Figure 2B).
Fitting the IC50 values versus the enzyme concentrations gives
a straight line (r2 = 0.9815) with a slope of 0.49, supporting the
idea that 4 stoichiometrically binds to TrxR. The selective
inhibition of WT TrxR but not U498C TrxR or GR demo-
nstrates that the Sec residue is crucial for the action of 4. To
further confirm the importance of the vicinal dithiol in binding
4, we generated the truncated TrxR (T-TrxR) by deleting the
C-terminal -Sec-Gly dipeptide. After preparing fluorophore-
labeled 2 (12) and fluorophore-labeled 4 (13, Scheme 2), we
performed a protein-labeling assay (Figure 2C). Both U498C
TrxR and T-TrxR could be efficiently labeled by 12 (lanes 2
and 4 in Figure 2C). However, 13 could bind only selectively
to U498C TrxR but not to T-TrxR (lane 1 in Figure 2C), a
protein having no accessible vicinal dithiol. This observation,
together with the potent inhibition of TrxR by 4, indicates that
4 specifically interacts with the C-terminal redox center of
TrxR. In addition, no apparent inhibition of Trx by 4, but remark-
able inhibition by 2, was observed (Figure 2D). Coincubation of
1 mM GSH with 80 nM TrxR could not prevent inhibition of
TrxR by 40 nM 4 (data not shown). Taken together, 4
selectively inhibits TrxR in vitro, and this inhibition is heavily
relies on the Sec residue and the vicinal dithiol/selenol motif in
the enzyme.
2.4. Interaction of 4 with TrxR in Cells. Treatment of
HL-60 cells with 4 causes a remarkable decrease of cellular
TrxR activity, with an IC50 value of around 1.4 μM, which is
3-fold lower than the value for 2 (4.0 μM, Figure 3A). 4 is the
most potent TrxR inhibitor in HL-60 cells among all of the
tested compounds (Figure S3), consistent with its highest cyto-
toxicity being toward HL-60 cells. Using our recently developed
TrxR probe, TRFS-green,32 inhibition of TrxR in living HL-60
cells was confirmed (Figure 3B,C). To further determine target-
ing TrxR by 4 in a cellular context, we performed a pulldown
assay. After incubating the cell lysate with immobilized 4
followed by DMPS elution, the TrxR1 protein was unambig-
uously detected (Figure 3D, lane b). Incubation of the lysate
with activated sepharose (A-Seph, Scheme 3) followed by the
same treatment does not give any signal (Figure 3D, lane c).
The cell lysate without any treatment was loaded as a positive
control (Figure 3D, lane a). Interestingly, exposure of HL-60
cells to 2 μM 4 for 12 h or 1 μM 4 for 24 h significantly
increased TrxR1 protein expression (Figure 3E), but the total
TrxR activity in the cells remained inhibited to less than 30% of
that of the control (data not shown). The elevation of the loss-
of-function enzyme (i.e., 4-modified TrxR) might contribute to
the ROS generation in cells (vide infra).
2.5. ROS Production in Vitro and in Cells. Several TrxR
inhibitors are known to modify the enzyme to shift it from
being an antioxidant to a source of ROS;34,36,43,44 thus, we
D
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2.6. Physiological Significance of Targeting TrxR by 4.
As we have demonstrated the selective interaction of 4 with
TrxR in vitro, we further asked the physiological relevance of
targeting TrxR by 4. Pretreatment of HL-60 cells with NAC,
a thiol antioxidant and GSH synthesis precursor in cells,
confers cytoprotection against 4-induced cell death, and higher
concentrations of NAC almost completely rescue the cells
(Figure 5A). 4-induced cell death was blocked by NAC, but
Figure 5. Physiological significance of targeting TrxR by 4. (A)
Protection of HL-60 cells by N-acetylcysteine (NAC). HL-60 cells
(2 × 104 cells) were incubated with the indicated concentrations of
NAC and 4 for 48 h. Cell viability was determined by the trypan blue
exclusion assay. (B) Augmentation of the cytotoxicity by inhibition of
GSH synthesis. HL-60 cells (1 × 104 cells) were treated with 100 μM
buthionine sulphoximine (BSO) for 24 h to lower the intracellular
GSH level, followed by 4 treatment for an additional 48 h. The
viability was determined by the MTT assay. (C) Cytotoxicity of 4 on
HEK-IRES and HEK-TrxR1 cells. The cells (1 × 104 cells) were
treated with the indicated concentrations of 4 for 48 h, and the cell
viability was determined by the MTT assay. (D) Cytotoxicity of 4 on
HeLa-shNT and HeLa-shTrxR1 cells. The cells (1 × 104 cells) were
treated with the indicated concentrations of 4 for 48 h, and the cell
viability was determined by the MTT assay. All data are expressed as
the mean SE of three experiments.
Figure 4. Elicitation of oxidative stress by 4. (A) Induction of
superoxide anions production by 4-modified TrxR in vitro. 4-modified
TrxR was incubated with NADPH, and cytochrome c was added to
monitor the production of superoxide anions. The inset shows the
change of absorbance at 550 nm after addition of cytochrome c and
superoxide dismutase (SOD). (B) Accumulation of ROS in the cells.
HL-60 cells were treated with 4 for 1 or 2 h followed by incubation
with DCFH-DA (10 μM) for an additional 30 min. Phase-contrast
(top) and fluorescence (bottom) images were acquired by
fluorescence microscopy (Leica DMI4000). Scale bar: 30 μm. (C)
Quantification of fluorescence intensity in individual cells in panel B.
The relative fluorescence intensity in individual cells was quantified
using the Leica Qwin software accompanied by the Leica microscope.
(D) Depletion of intracellular thiols by 4. After the HL-60 cells were
treated with 4, total cellular thiols were quantified by DTNB titration.
Data are expressed as the mean SE of three experiments.
other general antioxidants, including vitamin C and vitamin E,
did not prevent this process (data not shown), suggesting that
the induction of cell death by 4 might be related to GSH
depletion. Thus, we determined the effect of GSH on the cyto-
toxicity of 4. Consistent with the protection by NAC, inhibi-
tion of cellular GSH synthesis by BSO remarkably enhances
the cytotoxicity of 4 (Figure 5B). Under our experimental
conditions, pretreatment of HL-60 cells with 100 μM BSO for
24 h downregulates the intracellular GSH level to less than 20%
of that of the control (data not shown). GSH is a pivotal
component of the glutathione network, which is another redox
regulation system in cells besides the thioredoxin system, and
also acts as a backup of the thioredoxin system.45 Sensitizing
the cells to 4 by the depletion of GSH supports the involve-
ment of the thioredoxin system in the biological action of 4.
To further disclose the involvement of TrxR in the cyto-
toxicity of 4, we compared the sensitivity of HEK cells stably
overexpressing TrxR1 (HEK-TrxR1) and control cells that
were stably transfected with a vector (HEK-IRES) upon 4
treatment.33,46 As shown in Figure 5C, 4 displays significantly
higher cytotoxicity toward HEK-IRES cells than toward HEK-
TrxR1 cells. To further address the physiological relevance of
determined if 4 has a similar effect. As shown in Figure 4A,
4-modified TrxR displayed steady cytochrome c reduction
activity in the presence of NADPH, and this cytochrome c
reduction activity was inhibited by addition of SOD, indicating
the production of superoxide anions by such a process. To
extend this in vitro discovery, we also demonstrated the time-
dependent generation of ROS in HL-60 cells after 4 stimula-
tion (Figure 4B) by staining the cells with a ROS probe,
2′,7′-dichlorofluorescin diacetate (DCFH-DA).33,36 The rela-
tive ROS production is quantified in Figure 4C. Compound 4
not only inhibits the physiological function of TrxR but also
upregulats its expression in cells (Figure 3). As we demon-
strated earlier, 4-modified TrxR is still functional as a pro-
oxidant to oxidize NADPH and constantly generate ROS in
vitro; the elevation of the modified TrxR contributes, at least in
part, to the ROS accumulation in HL-60 cells. Cellular thiols are
endogenous antioxidants to neutralize ROS. As a consequence of
TrxR inhibition and ROS accumulation, a drastic loss of cellular
thiols was observed after treatment with 4 (Figure 4D).
E
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
TrxR-mediated 4 cytotoxicity, we generated a cell line stably
knocking down TrxR1 expression by transfection of shRNA
specifically targeting the enzyme. Because of the low trans-
fection efficiency of HL-60 cells, we chose HeLa cells for the
knockdown experiments.33,46 Importantly, 4 shows elevated
cytotoxicity toward HeLa-shTrxR1 cells compared to that of
the control cells (HeLa-shNT) (Figure 5D). The efficiency of
TrxR expression as well as the enzyme activity in TrxR-
overexpressing and knockdown cells is validated in Figure S5.
Collectively, our results strongly support the idea that the bio-
logical action of 4 in cells is related to its interaction with TrxR.
2.7. Induction of Apoptosis in HL-60 Cells. Abrogation
of apoptotic pathways is frequently found in malignant cells,
which arises from a complex interplay of genetic aberrations
and misregulated death pathways.47 Arsenic compounds are
known to activate apoptotic signaling in numerous types of
cells.48−51 Herein, we also demonstrated that 4 kills HL-60 cells
predominantly through the induction of apoptosis. The results
from the Annexin-V−FITC/PI double staining (Figure 6A,B)
indicate that 4 triggers apoptotic cell death in a manner that is
both dose- and time-dependent. Under all circumstances in our
experiments, the population of necrotic cells (FITC-negative
and PI-positive cells) was always as low as that of the control.
Caspase-3 is a crucial component of the apoptotic machinery
in different cell types, and the activation of caspase-3 is a central
event in the process of apoptosis.52 Thus, we determined the
cellular activity of caspase-3 after cells were treated with 4.
Compound 4 treatment significantly increased caspase-3
activity in HL-60 cells (Figure 6C). When HL-60 cells were
incubated with 4 followed by Hoechst staining, the majority
of the cells displayed condensed and highly fluorescent nuclei,
a characteristic morphology of cells undergoing apoptosis
(Figure 6D). Taken together, our data reveal that 4 mainly
induces apoptotic cell death in HL-60 cells.
The biological importance of inhibition of TrxR is at least
3-fold. First, the inhibition of TrxR reduces the availability
of reduced Trx, leading to a decrease in the activity of many
antioxidant enzyme systems that rely on Trx as an electron
donor, which eventually results in an accumulation of ROS and
the collapse of the cellular redox balance.53 Second, reduced Trx
directly interacts with various apoptosis-related enzymes, such
as ASK1,54 procaspase 3,55 and NF-κB,56 to suppress apoptosis;
thus, inhibition of TrxR would be expected to promote
apoptosis. Third, TrxR inhibition could result in the formation
of selenium compromised thioredoxin reductase-derived
apoptotic proteins (SecTRAPs),57,58 in which the Sec residue
in the active site is modified by electrophiles. These SecTRAPs
lose their physiological function (i.e., the ability to reduce
oxidized Trx), but they maintain NADPH oxidase activity for
the generation of ROS, which finally contributes to increased
intracellular oxidative stress. The elevation of loss-of-function
TrxR1 by 4 might lead to more SecTRAPs in HL-60 cells.
Taken together, inhibition of TrxR by 4 thus leads to oxidative
stress and promotes apoptosis.
Figure 6. Induction of apoptosis in HL-60 cells. (A) Analysis of
apoptosis by Annexin-V/PI double-staining assay. The cells show four
different populations marked as follows: double-negative (unstained)
cells are live cells (lower left, Q3), Annexin-V-positive and PI-negative
cells are in early apoptosis (lower right, Q4), Annexin-V/PI double-
stained cells are in late apoptosis (upper right, Q2), and PI-positive and
Annexin-V-negative cells are necrotic (upper left, Q1). (B) Quantifica-
tion of apoptosis by Annexin-V/PI double-staining assay. Cells in Q1
and Q3 were considered necrotic and live, respectively. The cells in Q2
and Q4 were considered apoptotic. (C) Activation of caspase-3 by 4.
HL-60 cells were incubated with the indicated concentrations of 4 for
24 h, and cellular caspase-3 activity was determined by a colorimetric
assay. All data are expressed as the mean SE of three experiments. (D)
Analysis of apoptosis by nuclear condensation. Hoechst 33342 staining
showed typical apoptotic morphology changes after treatment with 4.
HL-60 cells were incubated with different concentrations of 4 for 24 h
followed by Hoechst 33342 staining. Phase-contrast (top panel) and
fluorescence (bottom panel) images were acquired by fluorescence
microscopy.
oxidability hinder its bioavailability. Introducing the dithiol
moiety might be an efficient way to improve these draw-
backs.29,37,65 Furthermore, the dithiol ligands also contribute to
increasing the specificity of the drugs, as 2 nonselectively binds
to vicinal dithiols or thiol/selenols, whereas 4 appears to be
higher in affinity than 2. The cytotoxicity of PAO−dithiol com-
plexes is also dependent on dithiol ligands: the six-membered
dithiaarsane (4) gives stronger potency than the five-membered
one (3). Collectively, our preliminary structure−activity relation-
ship results may shed light on modifying melarsoprol and GSAO,
two organic arsenicals currently under clinical trials, to achieve
better efficacy. The observation that dithiol compounds at low
concentrations increase arsenite toxicity66 could also be explained
by our discoveries: dithiols complex with arsenites to improve
the bioavailability and the specificity, thus enhancing the toxicity.
Because of the high affinity of transition metals with thiol/
selenol, a vast amount of transition metal complexes have been
developed as TrxR inhibitors.21−23,59−64 These complexes usually
display high potency in the inhibition of TrxR in vitro. The small
organic molecule 4 quantitatively binds to TrxR, yielding com-
parable or superior potency to that of those metal complexes.
Despite the toxicity, arsenicals remain of high interest in the
treatment of various diseases.6,8,37 Arsenoxide is the active form
of most trivalent arsenicals. However, the high polarity and easy
F
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Although ATO has demonstrated great achievements in the
treatment of APL, clinical outcomes of this inorganic drug in
other tumors have been poor, mainly due to limited bioavailabi-
lity and severe side effects of the drug.2,8,67 We demonstrated
that 4 shows better potency than ATO in the present study and
clarified the mechanism underlying the anticancer activity of 4.
Furthermore, 4 is stable in cell culture medium or phosphate
buffered saline, and no significant loss of 4 was observed after
12 h (Figures S6 and S7). We expect that 4 could be a novel
cancer chemotherapeutic agent for further development.
selenocysteine; SecTRAPs, selenium compromised thioredoxin
reductase-derived apoptotic proteins; SOD, superoxide dis-
mutase; Trx, thioredoxin; TrxR, thioredoxin reductase
REFERENCES
■
(1) Beauchamp, E. M.; Uren, A. A new era for an ancient drug:
arsenic trioxide and hedgehog signaling. Vitam. Horm. 2012, 88, 333−
354.
(2) Evens, A. M.; Tallman, M. S.; Gartenhaus, R. B. The potential of
arsenic trioxide in the treatment of malignant disease: past, present,
and future. Leuk. Res. 2004, 28, 891−900.
(3) Waxman, S.; Anderson, K. C. History of the development of
arsenic derivatives in cancer therapy. Oncologist 2001, 6, 3−10.
(4) Zhang, T. D. Treatment of acute granulocytic leukemia with “Ai
Ling No. 1” − clinical analysis and experimental research. Zhongxiyi
Jiehe Zazhi 1984, 4, 19−20.
3. CONCLUSIONS
We discovered and fully elucidated the anticancer mechanism
of organoarsenic compound 4. 4 potently and selectively
inhibits TrxR via stoichiometrically binding to the C-terminal
Sec/Cys pair in the enzyme in vitro. The molecular mechanism
of 4 in cells involves upregulation but inhibition of TrxR,
elevation of ROS, depletion of cellular thiols, and eventual
induction of oxidative stress-mediated apoptosis. Knockdown
of TrxR enhances the cytotoxicity of 4, whereas overexpression
of the functional enzyme confers cytoprotection, providing
physiological significance to the interaction of TrxR with 4 in
cells. The novel targeting mechanism and the simplicity of
the structure with an easily manipulated amine group make 4
a promising scaffold for further development in discovering
potential cancer chemotherapeutic agents.
(5) Zhang, T. D.; Chen, G. Q.; Wang, Z. G.; Wang, Z. Y.; Chen, S. J.;
Chen, Z. Arsenic trioxide, a therapeutic agent for APL. Oncogene 2001,
20, 7146−7153.
(6) Miodragovic, E. U.; Quentzel, J. A.; Kurutz, J. W.; Stern, C. L.;
Ahn, R. W.; Kandela, I.; Mazar, A.; O’Halloran, T. V. Robust structure
and reactivity of aqueous arsenous acid-platinum(II) anticancer
complexes. Angew. Chem., Int. Ed. 2013, 52, 10749−10752.
(7) Chen, H.; Pazicni, S.; Krett, N. L.; Ahn, R. W.; Penner-Hahn, J.
E.; Rosen, S. T.; O’Halloran, T. V. Coencapsulation of arsenic- and
platinum-based drugs for targeted cancer treatment. Angew. Chem., Int.
Ed. 2009, 48, 9295−9299.
(8) Dilda, P. J.; Hogg, P. J. Arsenical-based cancer drugs. Cancer
Treat. Rev. 2007, 33, 542−564.
(9) Swindell, E. P.; Hankins, P. L.; Chen, H.; Miodragovic, D. U.;
O’Halloran, T. V. Anticancer activity of small-molecule and nano-
particulate arsenic(III) complexes. Inorg. Chem. 2013, 52, 12292−
12304.
(10) Arner, E. S.; Holmgren, A. Physiological functions of
thioredoxin and thioredoxin reductase. Eur. J. Biochem. 2000, 267,
6102−6109.
(11) Lu, J.; Holmgren, A. Thioredoxin system in cell death
progression. Antioxid. Redox Signaling 2012, 17, 1738−1747.
(12) Rigobello, M. P.; Bindoli, A. Mitochondrial thioredoxin
reductase purification, inhibitor studies, and role in cell signaling.
Methods Enzymol. 2010, 474, 109−122.
(13) Arner, E. S. Focus on mammalian thioredoxin reductases−
important selenoproteins with versatile functions. Biochim. Biophys.
Acta 2009, 1790, 495−526.
(14) Laurent, T. C.; Moore, E. C.; Reichard, P. Enzymatic synthesis
of deoxyribonucleotides. IV. Isolation and characterization of
thioredoxin, the hydrogen donor from Escherichia coli B. J. Biol.
Chem. 1964, 239, 3436−3444.
(15) Chae, H. Z.; Chung, S. J.; Rhee, S. G. Thioredoxin-dependent
peroxide reductase from yeast. J. Biol. Chem. 1994, 269, 27670−27678.
(16) Brot, N.; Weissbach, L.; Werth, J.; Weissbach, H. Enzymatic
reduction of protein-bound methionine sulfoxide. Proc. Natl. Acad. Sci.
U.S.A. 1981, 78, 2155−2158.
(17) Gonzalez Porque, P.; Baldesten, A.; Reichard, P. The
involvement of the thioredoxin system in the reduction of methionine
sulfoxide and sulfate. J. Biol. Chem. 1970, 245, 2371−2374.
(18) Powis, G.; Kirkpatrick, D. L. Thioredoxin signaling as a target
for cancer therapy. Curr. Opin. Pharmacol. 2007, 7, 392−397.
(19) Urig, S.; Becker, K. On the potential of thioredoxin reductase
inhibitors for cancer therapy. Semin. Cancer Biol. 2006, 16, 452−465.
(20) Biaglow, J. E.; Miller, R. A. The thioredoxin reductase/
thioredoxin system: novel redox targets for cancer therapy. Cancer.
Biol. Ther. 2005, 4, 6−13.
ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthetic procedures and characterization of organo-
arsenical compounds, experimental procedures for recombinant
proteins preparation, cytotoxicity evaluation, enzyme activity
assays, pull-down assay, western blotting, ROS detection,
cellular thiols measurement, and apoptosis assays. This material
is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: fangjg@lzu.edu.cn; Tel: +86 931-8912500; Fax: +86
931 8915557.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from the National Natural Science
Foundation of China (21002047), the Ministry of Education
of China (20100211110027), Lanzhou University (the
Fundamental Research Funds for the Central Universities,
lzujbky-2014-56), the National Natural Science Foundation of
China for Fostering Talents in Basic Research (J1103307), and
the 111 Project are gratefully acknowledged. The authors also
express heartfelt appreciation to Prof. Arne Holmgren at
Karolinska Institute for the recombinant rat TrxR and to Prof.
Constantinos Koumenis from the University of Pennsylvania
School of Medicine for shRNA plasmids as well as HEK-TrxR1
and HEK-IRES cells.
ABBREVIATIONS USED
■
(21) Cai, W.; Zhang, L.; Song, Y.; Wang, B.; Zhang, B.; Cui, X.; Hu,
G.; Liu, Y.; Wu, J.; Fang, J. Small molecule inhibitors of mammalian
thioredoxin reductase. Free Radical Biol. Med. 2012, 52, 257−265.
(22) Sun, R. W.; Lok, C. N.; Fong, T. T.; Li, C. K.; Yang, Z. F.; Zou,
T.; Siu, A. F.; Che, C. M. A dinuclear cyclometalated gold(III)−
APL, acute promyelocytic leukemia; BSO, buthionine
sulphoximine; DCFH-DA, 2′,7′-dichlorofluorescin diacetate;
GPx, glutathione peroxidase; GR, glutathione reductase;
NAC, N-acetylcysteine; ROS, reactive oxygen species; Sec,
G
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
phosphine complex targeting thioredoxin reductase inhibits hepato-
cellular carcinoma in vivo. Chem. Sci. 2013, 4, 1979−1988.
(23) Oehninger, L.; Kuster, L. N.; Schmidt, C.; Munoz-Castro, A.;
Prokop, A.; Ott, I. A chemical-biological evaluation of rhodium(I) N-
heterocyclic carbene complexes as prospective anticancer drugs.
Chem.Eur. J. 2013, 19, 17871−17880.
inorganic and methylated arsenicals in rat and human cells. Arch.
Toxicol. 2000, 74, 289−299.
(42) Nakamuro, K.; Sayato, Y. Comparative studies of chromosomal
aberration induced by trivalent and pentavalent arsenic. Mutat. Res.
1981, 88, 73−80.
(43) Fang, J.; Lu, J.; Holmgren, A. Thioredoxin reductase is
irreversibly modified by curcumin: a novel molecular mechanism for
its anticancer activity. J. Biol. Chem. 2005, 280, 25284−25290.
(44) Arner, E. S.; Bjornstedt, M.; Holmgren, A. 1-Chloro-2,4-
dinitrobenzene is an irreversible inhibitor of human thioredoxin
reductase. Loss of thioredoxin disulfide reductase activity is
accompanied by a large increase in NADPH oxidase activity. J. Biol.
Chem. 1995, 270, 3479−3482.
(45) Du, Y.; Zhang, H.; Lu, J.; Holmgren, A. Glutathione and
glutaredoxin act as a backup of human thioredoxin reductase 1 to
reduce thioredoxin 1 preventing cell death by aurothioglucose. J. Biol.
Chem. 2012, 287, 38210−38219.
(46) Javvadi, P.; Hertan, L.; Kosoff, R.; Datta, T.; Kolev, J.; Mick, R.;
Tuttle, S. W.; Koumenis, C. Thioredoxin reductase-1 mediates
curcumin-induced radiosensitization of squamous carcinoma cells.
Cancer Res. 2010, 70, 1941−1950.
(47) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next
generation. Cell 2011, 144, 646−674.
(48) Eguchi, R.; Fujimori, Y.; Takeda, H.; Tabata, C.; Ohta, T.;
Kuribayashi, K.; Fukuoka, K.; Nakano, T. Arsenic trioxide induces
apoptosis through JNK and ERK in human mesothelioma cells. J. Cell.
Physiol. 2011, 226, 762−768.
(49) Liu, L.; Trimarchi, J. R.; Navarro, P.; Blasco, M. A.; Keefe, D. L.
Oxidative stress contributes to arsenic-induced telomere attrition,
chromosome instability, and apoptosis. J. Biol. Chem. 2003, 278,
31998−32004.
(50) Bode, A.; Dong, Z. Apoptosis induction by arsenic: mechanisms
of action and possible clinical applications for treating therapy-resistant
cancers. Drug Resist. Updates 2000, 3, 21−29.
(24) Millet, R.; Urig, S.; Jacob, J.; Amtmann, E.; Moulinoux, J. P.;
Gromer, S.; Becker, K.; Davioud-Charvet, E. Synthesis of 5-nitro-2-
furancarbohydrazides and their cis-diamminedichloroplatinum com-
plexes as bitopic and irreversible human thioredoxin reductase
inhibitors. J. Med. Chem. 2005, 48, 7024−7039.
(25) Klossowski, S.; Muchowicz, A.; Firczuk, M.; Swiech, M.; Redzej,
A.; Golab, J.; Ostaszewski, R. Studies toward novel peptidomimetic
inhibitors of thioredoxin-thioredoxin reductase system. J. Med. Chem.
2012, 55, 55−67.
(26) Shen, S.; Li, X. F.; Cullen, W. R.; Weinfeld, M.; Le, X. C. Arsenic
binding to proteins. Chem. Rev. 2013, 113, 7769−7792.
(27) Sapra, A.; Thorpe, C. An arsenical-maleimide for the generation
of new targeted biochemical reagents. J. Am. Chem. Soc. 2013, 135,
2415−2418.
(28) Park, D.; Don, A. S.; Massamiri, T.; Karwa, A.; Warner, B.;
MacDonald, J.; Hemenway, C.; Naik, A.; Kuan, K. T.; Dilda, P. J.;
Wong, J. W.; Camphausen, K.; Chinen, L.; Dyszlewski, M.; Hogg, P. J.
Noninvasive imaging of cell death using an Hsp90 ligand. J. Am. Chem.
Soc. 2011, 133, 2832−2835.
(29) Huang, C.; Yin, Q.; Zhu, W.; Yang, Y.; Wang, X.; Qian, X.; Xu,
Y. Highly selective fluorescent probe for vicinal-dithiol-containing
proteins and in situ imaging in living cells. Angew. Chem., Int. Ed. 2011,
50, 7551−7556.
(30) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Specific covalent
labeling of recombinant protein molecules inside live cells. Science
1998, 281, 269−272.
(31) Lu, J.; Chew, E. H.; Holmgren, A. Targeting thioredoxin
reductase is a basis for cancer therapy by arsenic trioxide. Proc. Natl.
Acad. Sci. U.S.A. 2007, 104, 12288−12293.
(32) Zhang, L.; Duan, D.; Liu, Y.; Ge, C.; Cui, X.; Sun, J.; Fang, J.
Highly selective off-on fluorescent probe for imaging thioredoxin
reductase in living cells. J. Am. Chem. Soc. 2014, 136, 226−233.
(33) Duan, D.; Zhang, B.; Yao, J.; Liu, Y.; Sun, J.; Ge, C.; Peng, S.;
Fang, J. Gambogic acid induces apoptosis in hepatocellular carcinoma
SMMC-7721 cells by targeting cytosolic thioredoxin reductase. Free
Radical Biol. Med. 2014, 69, 15−25.
(34) Cai, W.; Zhang, B.; Duan, D.; Wu, J.; Fang, J. Curcumin
targeting the thioredoxin system elevates oxidative stress in HeLa cells.
Toxicol. Appl. Pharmacol. 2012, 262, 341−348.
(51) Chen, G. Q.; Zhu, J.; Shi, X. G.; Ni, J. H.; Zhong, H. J.; Si, G. Y.;
Jin, X. L.; Tang, W.; Li, X. S.; Xong, S. M.; Shen, Z. X.; Sun, G. L.; Ma,
J.; Zhang, P.; Zhang, T. D.; Gazin, C.; Naoe, T.; Chen, S. J.; Wang, Z.
Y.; Chen, Z. In vitro studies on cellular and molecular mechanisms of
arsenic trioxide (As2O3) in the treatment of acute promyelocytic
leukemia: As2O3 induces NB4 cell apoptosis with downregulation of
Bcl-2 expression and modulation of PML-RAR alpha/PML proteins.
Blood 1996, 88, 1052−1061.
(52) Budihardjo, I.; Oliver, H.; Lutter, M.; Luo, X.; Wang, X.
Biochemical pathways of caspase activation during apoptosis. Annu.
Rev. Cell Dev. Biol. 1999, 15, 269−290.
(35) Cai, W.; Zhang, L.; Song, Y.; Zhang, B.; Cui, X.; Hu, G.; Fang, J.
3,4,4′-Trihydroxy-trans-stilbene, an analogue of resveratrol, is a potent
antioxidant and cytotoxic agent. Free Radical Res. 2011, 45, 1379−
1387.
(36) Duan, D.; Zhang, B.; Yao, J.; Liu, Y.; Fang, J. Shikonin targets
cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in
human promyelocytic leukemia HL-60 cells. Free Radical Biol. Med.
2014, 70, 182−193.
(37) Loiseau, P. M.; Lubert, P.; Wolf, J. G. Contribution of dithiol
ligands to in vitro and in vivo trypanocidal activities of dithiaarsanes
and investigation of ligand exchange in an aqueous solution.
Antimicrob. Agents Chemother. 2000, 44, 2954−2961.
(53) Nordberg, J.; Arner, E. S. Reactive oxygen species, antioxidants,
and the mammalian thioredoxin system. Free Radical Biol. Med. 2001,
31, 1287−1312.
(54) Saitoh, M.; Nishitoh, H.; Fujii, M.; Takeda, K.; Tobiume, K.;
Sawada, Y.; Kawabata, M.; Miyazono, K.; Ichijo, H. Mammalian
thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase
(ASK) 1. EMBO J. 1998, 17, 2596−2606.
(55) Mitchell, D. A.; Morton, S. U.; Fernhoff, N. B.; Marletta, M. A.
Thioredoxin is required for S-nitrosation of procaspase-3 and the
inhibition of apoptosis in Jurkat cells. Proc. Natl. Acad. Sci. U.S.A. 2007,
104, 11609−11614.
(56) Matthews, J. R.; Wakasugi, N.; Virelizier, J. L.; Yodoi, J.; Hay, R.
T. Thioredoxin regulates the DNA binding activity of NF-kappa B by
reduction of a disulphide bond involving cysteine 62. Nucleic Acids Res.
1992, 20, 3821−3830.
(57) Anestal, K.; Prast-Nielsen, S.; Cenas, N.; Arner, E. S. Cell death
by SecTRAPs: thioredoxin reductase as a prooxidant killer of cells.
PLoS One 2008, 3, e1846.
(58) Anestal, K.; Arner, E. S. Rapid induction of cell death by
selenium-compromised thioredoxin reductase 1 but not by the fully
active enzyme containing selenocysteine. J. Biol. Chem. 2003, 278,
15966−15972.
(59) Oehninger, L.; Kuster, L. N.; Schmidt, C.; Munoz-Castro, A.;
Prokop, A.; Ott, I. A chemical−biological evaluation of rhodium(I) N-
(38) Stevenson, K. J.; Hale, G.; Perham, R. N. Inhibition of pyruvate
dehydrogenase multienzyme complex from Escherichia coli with mono-
and bifunctional arsenoxides. Biochemistry 1978, 17, 2189−2192.
(39) Heredia-Moya, J.; Kirk, K. L. An improved synthesis of arsenic−
biotin conjugates. Bioorg. Med. Chem. 2008, 16, 5743−5746.
(40) Sundberg, L.; Porath, J. Preparation of adsorbents for biospecific
affinity chromatography. I. Attachment of group-containing ligands to
insoluble polymers by means of bifuctional oxiranes. J. Chromatogr.
1974, 90, 87−98.
(41) Styblo, M.; Del Razo, L. M.; Vega, L.; Germolec, D. R.;
LeCluyse, E. L.; Hamilton, G. A.; Reed, W.; Wang, C.; Cullen, W. R.;
Thomas, D. J. Comparative toxicity of trivalent and pentavalent
H
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
heterocyclic carbene complexes as prospective anticancer drugs.
Chem.Eur. J. 2013, 19, 17871−17880.
(60) Wang, Y.; Liu, M.; Cao, R.; Zhang, W.; Yin, M.; Xiao, X.; Liu,
Q.; Huang, N. A soluble bis-chelated gold(I) diphosphine compound
with strong anticancer activity and low toxicity. J. Med. Chem. 2013,
56, 1455−1466.
(61) Koster, S. D.; Alborzinia, H.; Can, S.; Kitanovic, I.; Wolfl, S.;
Rubbiani, R.; Ott, I.; Riesterer, P.; Prokop, A.; Merza, K.; Metzler-
Nolte, N. A spontaneous gold(I)-azide alkyne cycloaddition reaction
yields gold-peptide bioconjugates which overcome cisplatin resistance
in a p53-mutant cancer cell line. Chem. Sci. 2012, 3, 2062−2072.
(62) Hickey, J. L.; Ruhayel, R. A.; Barnard, P. J.; Baker, M. V.;
Berners-Price, S. J.; Filipovska, A. Mitochondria-targeted chemo-
therapeutics: the rational design of gold(I) N-heterocyclic carbene
complexes that are selectively toxic to cancer cells and target protein
selenols in preference to thiols. J. Am. Chem. Soc. 2008, 130, 12570−
12571.
(63) Meyer, A.; Bagowski, C. P.; Kokoschka, M.; Stefanopoulou, M.;
Alborzinia, H.; Can, S.; Vlecken, D. H.; Sheldrick, W. S.; Wolfl, S.; Ott,
I. On the biological properties of alkynyl phosphine gold(I)
complexes. Angew. Chem., Int. Ed. 2012, 51, 8895−8899.
(64) Citta, A.; Schuh, E.; Mohr, F.; Folda, A.; Massimino, M. L.;
Bindoli, A.; Casini, A.; Rigobello, M. P. Fluorescent silver(I) and
gold(I)-N-heterocyclic carbene complexes with cytotoxic properties:
mechanistic insights. Metallomics 2013, 5, 1006−1015.
(65) Craig, S.; Gao, L.; Lee, I.; Gray, T.; Berdis, A. J. Gold-containing
indoles as anticancer agents that potentiate the cytotoxic effects of
ionizing radiation. J. Med. Chem. 2012, 55, 2437−2451.
(66) Jan, K. Y.; Wang, T. C.; Ramanathan, B.; Gurr, J. R. Dithiol
compounds at low concentrations increase arsenite toxicity. Toxicol.
Sci. 2006, 90, 432−439.
(67) Liu, B.; Pan, S.; Dong, X.; Qiao, H.; Jiang, H.; Krissansen, G. W.;
Sun, X. Opposing effects of arsenic trioxide on hepatocellular
carcinomas in mice. Cancer Sci. 2006, 97, 675−681.
I
dx.doi.org/10.1021/jm500221p | J. Med. Chem. XXXX, XXX, XXX−XXX